Project Summary Chronic stress is associated with enhanced abdominal pain (visceral hyperalgesia) in animal models and the human. Both animal models and humans demonstrate significant heterogeneity in their perception of pain in response to chronic stress, e.g. some individuals have a modest enhancement and others a marked enhancement in their perception of pain. The mechanistic basis for this diverse pain response to stress is unknown but represents one of the fundamentally important unanswered questions in basic and clinical pain research. Several hypotheses have been proposed to explain chronic stress-induced visceral hyperalgesia including sensitization of central and peripheral pain pathways and, recently, we reported that the magnitude of visceral pain response correlates with the magnitude of increased intestinal epithelial paracellular permeability linked to down-regulation of epithelial tight junction protein expression in the rat colon. Emerging evidence implicates a potentially pivotal role for epigenetic and endocannabinoid pathways in regulating pain perception. It is unknown whether epigenetic regulatory pathways play a role in chronic stress-induced increase in intestinal permeability and concomitant visceral hyperalgesia. We propose the novel hypothesis that the heterogeneous visceral pain response to chronic stress will be linked to distinct profiles of DNA methyltransferases (Dnmts) and histone tail modification enzymes observed in control rats that regulate intestinal epithelial tight junction protein expression, which will predict the magnitude of visceral hyperalgesia response to chronic stress. Specific Aim 1 will profile the visceral pain response to colorectal distention, colon permeability and epithelial tight junction protein expression in male Wistar rats before and after ten days of chronic, intermittent water avoidance stress. Each animal will serve as its own control. Specific Aim 2 will elucidate the epigenetic and endocannabinoid regulatory pathways that underlie the differential pain response to chronic stress including differential down-regulation of colon epithelial tight junction proteins, differential increase in paracellular permeability and higher visceral pain response in a subpopulation of male Wistar rats. To assess translatability of the animal results to the human, we will perform complementary studies using human colonoids derived from controls and age-matched patients with diarrhea-prone Irritable Bowel Syndrome (IBS-D) and differentiated Caco-2 cells exposed to stress levels of cortisol to measure permeability, tight junction protein expression and relevant epigenetic regulatory pathways before and after cortisol treatment. These studies will generate essential data for a subsequent R01 application to explore the mechanistic basis for the heterogeneous pain response to stress.